SUMMARY: RNA splicing plays a central role in the generation of proteome diversity and in gene regulation. Splicing impacts vital cellular processes, such as cell-fate and differentiation, acquisition of tissue-identity, and organ development. Thus, defects in splicing has been linked to many diseases, including spinal muscular atrophy, Duchenne muscular dystrophy, Parkinson?s disease, and several types of cancer. RNA splicing is even more relevant in cancer due to the higher incidence of mis-splicing events than in normal cells. Thus, it is not surprising that aberrant splicing has been linked to important hallmarks of cancer such as proliferation, proliferation, apoptosis evasion, metastasis, and angiogenesis, and in the development of cellular resistance against cancer therapeutics. Despite the significance of splicing in cancer and other diseases, drug discovery efforts targeting them are far and few. A critical bottleneck for such efforts is the lack of robust high-throughput assay tools to monitor endogenous spliced RNA in the cell. Even though there are excellent tools such as RT-qPCR and RNA- seq to study RNA splicing, they are not readily adaptable for high-throughput screening (HTS) due to their complex and time-consuming methodology. While splice-mini-gene method offers the advantage of higher- throughput, it lacks in the ability to monitor the endogenous target RNA due its artificial design. Thus, there is an unmet need for simple and robust HTS-ready assay tools to monitor RNA splicing. Addressing this need, we proposed the development of an easy-to-use, HTS-ready splice sensor platform that can specifically detect a spliced RNA isoform of interest. In the Phase I, we used pyruvate kinase isoforms M1 (PKM1) and M2 (PKM2) as model targets and developed prototype sensors that emit fluorescence signal when they bind to their respective target RNA isoforms. PKM isoform switching is one of the ways cancer cells reprogram their glycolytic pathways to meet cellular demands of energy and biosynthetic intermediates for growth and proliferation. With the goals of commercializing the splice sensor platform for drug discovery, in the Phase II, we will perform a pilot HTS for chemical modulators of PKM splicing and validate the splice sensor as a HTS-ready platform. To demonstrate the versatility of the assay platform to target any RNA isoform of interest, we will port the modular splice sensor platform to detect two new RNA isoforms linked to cancer. To operationalize the splice sensor development process in the Aim 3, we will streamline standard operating procedures and create benchmarks for the development, production, and kitting of the splice sensor assay. Lastly, we will create a next-generation splice sensor-origami (SSO) system that will comprise of a Spinach reader and a user-customizable companion probe set to target any spliced RNA isoform of interest. The SSO will rely on toe-hold mediated strand displacement to achieve specificity and activation of Spinach fluorescence. Thus, at the end of Phase II, we will commercialize the splice sensors developed as assay kits for direct sales and work with biopharma companies to create custom splice sensors for internal drug discovery needs.